1213 Original Article NATURAL OCCURRENCE OF Pasteuria nishizawae IN SOYBEAN AREAS OCORRÊNCIA NATURAL DE Pasteuria nishizawae EM ÁREAS DE SOJA Camilla Buiatti VICENTE 1; Maria Amelia dos SANTOS 2 1. Engenheira Agrônoma, Mestre em Fitopatologia pela Universidade Federal de Uberlândia - UFU, Syngenta, Uberlândia, MG, Brasil; 2. Professora, Doutora, Instituto de Ciências Agrárias – ICIAG - UFU, Uberlândia, MG, Brasil. ABSTRACT: Soybean ( Glycine max (L.) Merrill) is a widely grown crop of economic prominence. Brazil is considered as the second largest worldwide producer and exporter. Soybean cyst nematode, Heterodera glycines Ichinohe, in one of the most serious threats for this crop, considered as its most destructive parasite. The first report of this disease in Brazil was recorded during the 1991/1992 harvest. Control of nematodes is more challenging if compared with other plant diseases control. Thus, there is a growing demand to search for alternative control practices that will not harm the environment nor the human being. Therefore, the highly specific bacteria Pasteuria spp. Metchnikoff, represents an auspicious biological control agent against nematodes. The biological control of the soybean cyst nematode with the bacterium Pasteuria nishizawae Sayre has proven an excellent choice and has been studied by different scientists. The objective of this work was to determine the natural occurrence of Pasteuria nishizawae in Brazilian soils. The experiment was performed under greenhouse conditions at the Research unit from Syngenta® in Uberlandia-MG, Brazil, with soil samples originated from soybean planted areas from the Brazilian states of Bahia, Goiás, Mato Grosso and Paraná. Fractions of 150 cm 3 of soil were withdrawn to be processed by the centrifugal flotation technique in sucrose solution. Aliquots of 1 mL from the obtained suspension were observed in Peters’ chamber with the aid of an inverted light microscope, in order to verify the absence or presence of bacterial endospores attached to the cuticle of the nematodes extracted from the soil samples. The frequency of Pasteuria nishizawae incidence was of 100% within the samples analyzed. KEYWORDS: Bacteria, Biological control, Cyst nematode, Glycine max . INTRODUCTION In tropical and subtropical countries nematodes find ideal humidity and temperature Soybean is a crop of economic importance, conditions for reproduction and feeding. Thus, being the most planted oilseed in the world. Brazil is worsening the efficiency of control of these the second largest producer and exporter of soybean pathogens, that are very difficult to eradicate once in the world, with a planted area of approximately they are established within an area (TORRES et al., 26,4 to 27,3 million hectares and a production of 2008). about 80,08 to 82,99 million tons in 2012/2013 Biological control is an alternative method (CONAB, 2013). to control plant parasites, as nematodes. Biological The soybean cysts nematode (SCN), control of nematodes can be achieved by the use of Heterodera glycines , is one of the most severe plant appropriate fungi or bacteria. Predators and egg disease problems of this crop, considered as the parasites are certainly the most studied organism most destructive parasite of soybean plants (NOEL, and the best fitted as nematode’s biological control 1992). agents (JATALA, 1986; NORDBRING-HERTZ et In order to control these nematodes the al., 2002). primary common methods used are chemical control The first observation of Pasteuria in a plant (nematicides), the use of resistant varieties and crop parasite nematode, Pratylenchus pratensis de Man, rotation. Chemical control by means of nematicide was made by Thorne (1940) whom classified the application has proven expensive as well as organism as a Protozoa and named it as Dubosquia detrimental to the environment, to the human being, penetrans . However, studies performed by Mankau to the wild fauna and to the beneficial organism (1975), re-classified the organism as a prokaryote of within the soil. The use of resistant varieties is a the Genus Bacillus Cohn. The re-discovery of natural approach, highly recommended to control Pasteuria ramose by Sayre et al. (1977) and its plant pests and diseases. However, in the case of morphological similarities with Bacillus penetrans this specific nematode, the availability of resistant Mankau suggested that these two bacteria had a varieties for the farmer is scarce, due to the great common generic relation. In 1985, Sayre and Starr number of races or HG types of SCN occurring in named the organism that parasite Meloidogyne Brazil (FERRAZ; FREITAS, 2004). incognita as Pasteuria penetrans (Thorne) Sayre & Starr, as well as Pasteuria nishizawae is specific to Received: 21/01/16 Biosci. J., Uberlândia, v. 32, n. 5, p. 1213-1220, Sept./Oct. 2016 Accepted: 06/06/16 1214 Natural occurrence of Pasteuria nishizawae… VICENTE, C. B.; SANTOS, M. A. the soybean cysts nematode ( Heterodera glycines ), endospore and penetration of the germ tube inside Pasteuria thornei Sayre & Starr is specific to the the nematode begin soon after penetration of the nematode of the root lesions according to Stirling & J2’s in the radicular system. Otherwise, the Wachtel (1980), while Pasteuria usgae is specific to endospore does not germinate and consequently no the nematode Belonolaimus longicaudatus Rau infection by Pasteuria will arise. For this reason, according to Rau Giblin- Davis et al. (2003). observations based only on diseased cysts will result Pasteuria is a gram-positive bacterium that in an incomplete analysis of Pasteuria ’s life cycle, forms an endospore. These bacteria were found as which would explain why germination of P. parasites of many economically important nishizawae is not observed (SAYRE et al., 1991). nematodes (SAYRE; STARR 1988). The Genus After the endospore’s germination, primary Pasteuria spp. was structured from 323 nematode cauliflower-like colonies are formed within the J3. species belonging to 116 Genera, including plant Development of the bacterium was observed only in parasitic nematodes, entomopathogenic nematodes, female adults but not in males. Later, the bacterial predator nematodes and free living nematodes sporulation can be observed with the development (CHEN; DICKSON 1998). of a structure similar to a chunk of grapes, which The analysis of the 16S rRNA gene can be observed also in immature females of fourth sequences is a sensible and well-established tool for stage and cysts. Finally, the presence of mature the detection and phylogenetic analysis of bacteria sporangia and endospores can be observed, varying (STAHL, 1997). The use of sequences of the gene in number (30.000 to 820.000 with mean value and 16S rRNA may lead to identification of species of standard deviation of 314.00 and 234.000, Pasteuria and to evaluation of the diversity of these respectively) as a function of the size of the cyst or microorganisms within a population of nematodes’ female nematode (ATIBALENTJA et. al., 2004). samples, thus being a tool of a great utility for the Until now, P. penetrans is the most studied comprehension of the parasite-nematode species due to its potential as a biological control interactions and the ecology of Pasteuria (EBERT agent. However, technical hitches for mass et al., 1996; ANDERSON et al., 1999; production in vitro had made its commercial ATIBALENTJA et al., 2000; BEKAL et al., 2001). production problematic. In order to introduce All Pasteuria species known are obligate bacteria in the soil environment for field parasites. Until recently the Genus had four species experiments, dry roots’ powder infected by the Pasteuria nishizawae , P. penetrans , P. ramosa and bacteria was used, as proposed by Stirling and P. thornei (OOSTENDORP et al., 1990; Chen; Wachtel (1980). Dickson, 1998). Thus, the objective of the present work was For many years attempts to obtain an axenic to verify the natural occurrence of Pasteuria culture of Pasteuria spp. resulted in failure nishizawae Sayre in soils under soybean cultivation. (WILLIAMS et al., 1989; BISHOP; ELLAR, 1991). However, an advance towards the success to obtain MATERIAL AND METHODS an in vitro culture of P. penetrans was announced by Hewlett et al. (2002). Soil samples with cysts coming from A life cycle study of the bacterium that different established soybean growing areas from parasites the nematode Heterodera glycines was Brazil (Table 1), were processed by the centrifugal performed by means of germination of the flotation in sucrose solution technique (Jenkins, endospore that infects J2’s for production of a next 1964). generation endospores in adult females and cysts. Part of those ten processed soil samples Descriptions were based in microscopic were added, individually, to ceramic pots and examination of successive juvenile stages of H. preserved under greenhouse at Syngenta’s glycines excised from soybean roots, unlike P. experimental station in Uberlândia, MG – Brazil. nishizawae , that were based exclusively on the These pots were planted with the susceptible examination of diseased cysts (SAYRE et al., 1991). soybean cultivar ‘Lee 74E’, in order to promote According to Atibalentja et al. (2004), multiplication of Heterodera glycines , as shown in bacterium development, germination of the Figure 1. Biosci. J., Uberlândia, v. 32, n. 5, p. 1213-1220, Sept./Oct. 2016 1215 Natural occurrence of Pasteuria nishizawae… VICENTE, C. B.; SANTOS, M. A. Table 1. Place of origin of the samples analyzed in the essay for natural occurrence of Pasteuria nishizawae in Brazilian soils. Identification County State H.g 1 Jataí GO H.g 2 Sorriso MT H.g 3 Campo Alegre GO H.g 4 Lucas do Rio Verde MT H.g 5 Luís Eduardo Magalhães BA H.g 6 Campo Alegre GO H.g 7 Nova Mutum MT H.g 8 Campo Alegre GO H.g 9 Luís Eduardo Magalhães BA H.g 10 Londrina PR Figure 1. Populations of Heterodera glycines originated from field soil samples, being multiplied and preserved under greenhouse conditions. A soil aliquot of 150 cm 3 was obtained 28 presence or absence of endospores of the bacterium days after sowing and then processed by the attached to the nematode’s cuticle (Figure 2).